Carbachol inhibits the calcium-dependent action

Transcription

1 436 Pertussis Txin-Insensitive Phsphinsitide Hydrlysis, Membrane Deplarizatin, and Psitive Intrpic Effect f Carbachl in Chick Atria Tsunemi Tajima, Yasuhir Tsuji, Jan Heller Brwn, and Achilles J. Pappan Muscarinic agnists can stimulate rather than inhibit cardiac muscle in sme preparatins. In left atria frm hatched chicks, treatment with pertussis txin reversed the membrane actin f carbachl frm hyperplarizatin t deplarizatin and reversed the intrpic effect f carbachl frm negative t psitive. Acetylchline als deplarized the membrane and increased the frce f cntractin in atria frm pertussis-txin-treated chicks althugh xtremrine did nt. These chlinergic respnses were blcked by atrpine but nt by adrenceptr antagnists, suggesting that they are mediated via muscarinic receptrs and are nt due t actins f endgenusly released catechlamines. Muscarinic receptr stimulatin leads t tw distinct bichemical respnses in chick atria: inhibitin f adenylate cyclase and activatin f phsphinsitide (PI) hydrlysis. The frmer is lst in atria frm pertussis-txin-treated chicks, whereas the PI respnse persists. The pharmaclgic characteristics f the PI respnse resemble thse f the deplarizatin and psitive intrpic respnse. Bth are insensitive t blckade by pertussis txin, require high cncentratins f carbachl, and are elicited by acetylchline but nt by xtremrine. The present study suggests that muscarinic agnist-induced PI turnver may be respnsible fr the membrane deplarizatin and psitive intrpic effects f carbachl and acetylchline; that an increase in Na + cnductance underlies these respnses; and that it is stimulated either by an increase f intracellular calcium mbilized by insitl triphsphate and/r by activatin by prtein kinase C. (Circulatin Research 1987;61: ) Carbachl inhibits the calcium-dependent actin ptential and cntractins in the atrium by an actin n muscarinic receptrs (machr).' The machr are linked t the catalytic unit f adenylate cyclase by G i( a guanine nucletide binding prtein that transduces the receptr signal int inhibitin f enzymatic activity. The attendant reductin f cyclic adensine 3',5'mnphsphate (camp) synthesis and f the phsphrylatin f membrane cmpnents diminishes the entry f Ca 2+ thrugh vltagedependent channels. 2 " 4 In frg ventricular muscle, acetylchline inhibits the pening f Ca 2+ channels by activating a cyclic guansine mnphsphate (cgmp)- dependent phsphdiesterase that diminishes camp cntent. 5 Muscarinic receptrs are als linked t activatin f atrial K + channels thrugh a guanine nucletide binding prtein, apparently G, and/r G O. 6 ~ 9 Guanine nucletide binding prteins btained frm human erythrcytes 10 and frm bvine cerebral crtex" have been shwn t activate muscarinic K + channels directly in islated atrial cell membrane patches. Frm the Divisin f Pharmaclgy, Department f Medicine, Schl f Medicine, University f Califrnia at San Dieg, La Jlla, Calif., and the Department f Pharmaclgy, University f Cnnecticut Health Center, Farmingtn, Cnn. Supprted by USPHS grants HL (A.J.P.) and HL and HL (J.H.B.). Address fr reprints: A.J. Pappan, Department f Pharmaclgy, University f Cnnecticut Health Center, Farmingtn, CT Received December 12, 1986; accepted April 3, Althugh there is significant disagreement between these reprts cncerning the suitability f G (frm brain) and the rle f a/3y vs. fiy subunits as cmpnents f the reactin, the results substantiate the previusly advanced cnclusin that n secnd messenger need be invlved fr muscarinic agnists t pen K + channels. 7 " 9 Muscarinic agnists thereby increase a specific K + cnductance, hyperplarize the membrane, and decrease actin ptential duratin, 79 Acetylchline is prpsed t act n specific K + channels that are distinct frm backgrund K channels (i KI ) in sinatrial nde, atrial muscle, and Purkinje fibers. 12 " 15 The reduced influx f Ca 2+ and the increased efflux f K + bth cntribute t the negative intrpic actin f muscarinic agnists in atrial muscle. 1 Treatment f chicks with pertussis txin (islet activating prtein, IAP) under cnditins in which 90 t 95% f G, and G are ribsylated 16 prevents the inhibitin f adenylate cyclase and Ca 2+ entry, the activatin f K + channels, and the negative intrpic effect f carbachl. 9 Als, under these cnditins, carbachl stimulates rather than inhibits atrial cells by deplarizing the membrane and exerting a psitive intrpic actin. Because these stimulant effects are pertussis-txin-insensitive, they apparently ccur via muscarinic receptrs whse link t intracellular effectrs is independent f G ( and G,,.' 6 Carbachl stimulates the hydrlysis f phsphinsitides (PI) in embrynic chick atria and chick heart cells thrugh activatin f machr. l7 This effect is nt blcked by treatment f chick heart cells with pertussis

2 Tajima et al PI Hydrlysis and Muscarinic Stimulatin 437 txin. 18 These bservatins, tgether with thse cncerning deplarizatin and psitive intrpic actins f carbachl in the hatched chick atrium, prmpted the hypthesis that carbachl may be acting thrugh the PI cycle t regulate in permeability and frce develpment in a nvel manner that results in stimulatin. The studies reprted here describe the stimulant effects f muscarinic agnists n membrane ptentials and cntractins and test the hypthesis that these respnses are related t activatin f PI hydrlysis. Materials and Methds Pertussis Txin Treatment White Leghrn chicks (5 t 7 days after hatching) were injected intravenusly (jugular vein) either with 1.0 t 1.5 fxg f pertussis txin (List Bichemicals, Campbell, Calif.) r with an equal vlume (50 fi\) f 0.9% NaCl and hused in an incubatr at 35 C. After 2 t 3 days, the animals were decapitated, their hearts were remved, and the atria were retained fr experiments. Electrphysilgy and Cntractin Experiments Left atrial muscle frm pertussis txin r vehicletreated chicks was used fr electrphysilgy and cntractin experiments. Atrial muscle strips were superfused with aerated (95% 0,-5% CO 2 ) mdified Tyrde's slutin (ram cncentratins: Na , K + 5.4, Ca , Mg , Cl" 148, HC<V 11.9, H 2 P<V 0.4, and glucse 5.5) at 37 C. Membrane ptentials were recrded with standard micrelectrde techniques. Cells were impaled with glass micrelectrdes filled with 3 M KCI (tip resistance 15 t 30 Mfl). The membrane ptential was led thrugh a high input impedance preamplifier (WPI type 701, Wrld Precisin Instruments, New Haven, Cnn.) and was displayed n an scillscpe. A single sucrse gap methd was used t btain an estimate f changes in membrane resistance prduced by carbachl and t adjust the steady membrane ptential t desired values by passing cnstant current pulses thrugh the tissue. This experimental setup and its limitatins have been described in a reprt frm this labratry. l9 Assuming that internal lngitudinal resistance is cnstant, the change in electrnic ptential prduced by a drug in respnse t a cnstant current pulse yields an estimate f the rati f membrane resistance (AR) by the relatin (R.WR^J-ktAV^AV^,,). The assumptin f cnstant internal lngitudinal resistance has been challenged 20 because acetylchline (1.1 xlo" 5 M) increased the resistivity f the intercellular pathway between atrial cells by 25%. Changes in plarizatin resistance up t 29% at the end f 500-yum fiber bundles culd escape detectin by ur recrding prcedures, as previusly reprted. 19 Fr these reasns, it is pssible fr muscarinic agnists t increase membrane cnductance t Na + and yet nt be bserved, particularly when plarizatin resistance is lw befre drug additin. When membrane resistance is increased by additin f 20 mm Cs +, the afrementined limitatins are partially ffset, and a reductin in membrane resistance cnsistent with the ability f carbachl t increase membrane permeability t Na + has been measured. 2 ' (It is f interest that Krth and Kuhlkamp bserved n deplarizatin when carbachl increased intracellular Na + activity in the presence f nrmal Tyrde's slutin yet were able t detect a mv deplarizatin after membrane resistance had been increased by additin f Ba 2+ r the missin f K + frm the Tyrde's slutin.) Data were taken nly frm cells in which a stable impalement was maintained thrughut the experiment. Muscle cntractins were recrded with a frce displacement transducer (FT03C, Grass Instrument C., Quincy, Mass.) using a technique described previusly. 22 Left atrial strips (apprximately 1 X 4 mm) were set at a rest length that prvided maximum frce develpment. The muscles were paced by a punctate silver electrde (100 yum diameter) with rectangular vltage pulses (1.5 x threshld vltage, 0.5 millisecnd duratin, 3 Hz). Phsphinsitide Measurements Right and left atria frm untreated r pertussistxin-treated chicks were remved and labelled with 20 /LtCi/ml [ 3 H]insitl (New England Nuclear, Bstn, Mass.) fr 90 minutes in a bicarbnate buffered Krebs salt slutin gassed with 95% O 2-5% CO 2. After remving [ 3 H]insitl and replacing with fresh medium, drug (in 10 mm LiCl) was added fr 30 minutes. T end the assay, atria were bltted dry and frzen in Fren cled with liquid nitrgen. Frzen atria were hmgenized in 10% TCA and the pellet remved by centrifugatin in a Beckman micrfuge B (Smerset, N.J.) fr 60 secnds. The supernatant was washed 5 times with 4 vlumes f ice-cld, water-saturated ether t remve TCA. The sample was run n in-exchange clumns and the insitl phsphate ([ 3 H]InsP) cllected, as described elsewhere. 23 Data were nrmalized fr tissue prtein cntent, and assayed by the methd f Bradfrd. 24 Nrmalized data frm right and left atria were nt different and were therefre cmbined. Measurements are given as mean±sem. The statistical significance between sample means was estimated with Student's t test. Results Carbachl Deplarizatin f Atrial Membranes Frm Pertussis-Txin-Treated Chicks: Cncentratin and Agnist Dependence Carbachl (10~ 8 t 10" 6 M) hyperplarized the resting membrane in cells frm saline-treated animals. 8 As shwn in Figure 1A, a high cncentratin f carbachl (10" 4 M) evked a relatively small hyperplarizatin that tended t dissipate during expsure. As we suggested previusly, 16 the reduced magnitude f the carbachl-induced hyperplarizatin at 10~ 4 M is prbably the result f the cncmitant generatin f a deplarizatin. The ccurrence f carbachl-induced deplarizatin is seen when the drug-induced hyperplarizatin is prevented by treatment with pertussis txin (Figure IB). Carbachl deplarized the mem-

3 438 Circulatin Research Vl 61, N 3, September 1987 SALINE I. A. P. Carb IO" 5 M Carb IO"*M I Carb IO' 4 M + Atrpine Carb B I IO" 4 M Carb IO" 4 M + Alrpine 10 mv I min FIGURE 1. Effects f carbachl (10 " M) n resting ptential in atrial cells frm saline (A, A') and lap-treated (B,B') chicks. Carbachl hyperplarized membrane f atrial cell frm salinetreated chick (A); this effect was blcked by3xlo~'m atrpine (A')- Carbachl deplarized membrane f atrial cell frm IAF'-treated chick (B); atrpine als blcked this respnse (B ') Vltage and time calibratins apply t all recrds. Initial resting ptential was 80 mv in A,A' and 77 mv in B,B'. brane by 5 mv, and the deplarizatin was sustained thrughut the 2 minutes f supervisin with the drug. Atrpine (3 x 10~ 7 M) prevented carbachl frm evking hyperplarizatin (Figure 1A') and deplarizatin (Figure IB') f atrial cells frm saline- and pertussistxin treated animals, respectively. Pertussis txin treatment blcks the decrease f plarizatin resistance (Rp,,,), that is, the increase f K + cnductance elicited by 10~ 6 M carbachl. 9 Similar results were btained when 10" 4 M carbachl was tested in atria frm pertussis-txin-treated animals. Membrane plarizatin resistance (R^,) averaged 7.0 ± 1.8 Kft {n = 7) in unstimulated atrial cells befre additin f carbachl and 7.2 ±1.2 Kft in the presence f 10~ 4 M carbachl (p>0.1). (The failure t detect an increased membrane cnductance t Na + under these cnditins can be accunted fr by the experimental limitatins described in "Materials and Methds.") The cncentratin dependence fr the deplarizatin evked by carbachl is illustrated in Figure 2. This experiment was dne with agnist applicatin frm a pipette (50 /urn tip diameter) cntaining the desired drug cncentratin. The magnitude f the carbachlinduced deplarizatin under steady-state cnditins was the same if the drug was applied by superfusin. As shwn in Figure 2, deplarizatin amunted t 1 mv, 5 mv, and 6.3 mv fr 10" 5, 10" 4, and 10~ 3 M carbachl, respectively. Acetylchline als deplarized atrial membranes frm pertussis-txin-treated chicks, but xtremrine did nt. A representative experiment is shwn in Figure 3. Superfusin with carbachl (10~ 4 M) elicited a 5.5 mv deplarizatin while an equimlar cncentratin f acetylchline deplarized the membrane by 1.4 mv (Figure 3). Oxtremrine (10" 4 M) had n effect n the resting ptential f atrial cells. The results f all experiments in which these muscarinic agnists were tested are shwn in Figure 4. Clearly, carbachl had the lwest threshld (10~ 5 M) and evked the largest icr 4 M IO" 3 M i I m i i n i c mv «w FIGURE 2. Cncentratin dependence f carbachl-induced deplarizatin in atrial cell frm lap-treated chick. A 50-fxm diameter pipette cntaining given carbachl cncentratin in Tyrde's slutin was placed in bath within 1 mm f tissue, where it remained fr 25 secnds. Deplarizatin prduced by carbachl increased in amplitude and rate f initiatin as carbachl cncentratin increased frm I0'! t IO' J M. Initial resting ptential was 72 mvfr each recrd. Vltage and time calibratins apply t all recrds. deplarizatin (6.9±0.5 mv at 10" 3 M). Deplarizatin by acetylchline was nt significant belw a cncentratin f 10" 4 M. At 10" 3 M, the acetylchlineinduced deplarizatin (1.9±0.2 mv) was apprximately 25% f that evked by carbachl. Oxtremrine was remarkable insfar as it failed t deplarize atrial membranes frm pertussis-txin-treated chicks at any cncentratin frm 10~ 6 M t 10~ 3 M. Vltage Dependence Deplarizatin by carbachl was als influenced by membrane vltage. The single sucrse gap methd was used t set the membrane ptential at a desired value, and the amplitude f the deplarizatin by carbachl was measured. The results f all experiments (8 different tissues) are shwn in Figure 5. The deplarizatin induced by 10~ 4 M carbachl was maximal Carbachl IO" 4 M Acetylchline 10" M Oxtremrine IO' 4 M I min I5mv FIGURE 3. Agnist dependence fr muscarinic-induced deplarizatin. Recrds are frm 3 atrial cells frm as many lap-treated chicks. At arrws, superfusin was changed t ne cntaining carbachl, acetylchline, r xtremrine, all at 10'' M. Vltage and time calibratins apply t all recrds. Initial resting ptential befre drug additin was 72 mv (carbachl), -78 mv (acetylchline), and 79 mv (xtremrine).

4 Tajima et al PI Hydrlysis and Muscarinic Stimulatin 439 > a. CAHB4CH0L (n.6) ACETYLCHOLINE O D OXOTREMORINE < AGONIST (M) FIGURE 4. Summary f cncentratin dependence and agnist dependence fr deplarizatin f atrial cells frm IAP-treated chicks. Ordinate: deplarizatin amplitude in mv; abscissa: agnist cncentratin. Number f cells given in parentheses. (~4.7 mv) between -70 and -80 mv; the average resting ptential in these preparatins was 76 ±2 mv befre applicatin f current t change membrane vltage. At membrane ptentials mre negative than 90 mv and mre psitive than 50 mv, the amplitude f the carbachl-induced deplarizatin decreased. Because the multicellular preparatins used are subject t in depletin and accumulatin at hyperplarized and deplarized ptentials, respectively, a mre precise determinatin f the vltage dependence fr carbachl-induced deplarizatin will have t await vltage clamp experiments n islated atrial mycytes. Hwever, ur results fr the vltage dependence f carbachl-induced deplarizatin are similar t thse f thers wh have studied the vltage dependence f digitalis-induced transient inward currents in multicellular cardiac Purkinje fibers. In atrial cells frm saline-treated animals, carbachl (10~ 4 M) hyperplarized the resting membrane by almst 4 mv (Table 1). The reversal ptential (E rev ) fr carbachl was 84 ±2.3 mv (Table 1). Assuming 1 6 kj 5 CL s M a. < UJ TO MEMBRANE POTENTIAL (mv) FIGURE 5. Vltage-dependence f deplarizatin induced by carbachl (10'' M) in atrial cells frm IAP-treated chicks. Ordinate: deplarizatin amplitude in mv: abscissa: membrane ptential in mv. Number f cells is shwn in parentheses. Membrane ptential was set by cnstant current pulses applied thrugh tissue in single sucrse gap chamber. Carbachl was added t superfusin fluid, and drug-induced deplarizatin was measured at steady-state (2 minutes) in each instance. that the intracellular K + cncentratin in atria frm hatched chicks is the same (120 t 130 mm) as in atria frm 18-day embrys, 27 the estimated K + equilibrium ptential ranges frm 82.2 t 84.3 mv. Therefre, the results f these experiments in saline-treated chicks are cnsistent with the well-knwn ability f muscarinic agnists t increase a specific K + cnductance in atrial cells.' By cntrast, carbachl (10~ 4 M) deplarized the atrial membrane f pertussistxin-treated chicks by 5 ± 0.7 mv. The magnitude f the deplarizatin was reduced at vltages between 50 mv and 10 mv. The pssibility that there is a reversal ptential fr the carbachl-induced deplarizatin at vltages mre psitive than 10 mv culd nt be tested because the cntrl f membrane ptential at vltages less negative than 10 mv is uncertain. Lack f Effect f Tetrdtxin and Verapamil The deplarizatin induced by carbachl was nt significantly antagnized by either tetrdtxin (TTX, 3x 10" 7 M) r verapamil (10" 6 M). In 9 individual cells frm different preparatins, carbachl (10~ 4 M) deplarized the resting membrane by 4.2±0.2 mv in the absence f TTX and by 4.5 ±0.3 mv in the presence f TTX. Carbachl deplarized by 5.2 ±0.6 mv in the absence and by 5.2±0.5 mv in the presence f verapamil (n = 8). The tissues were expsed t either TTX r verapamil fr at least 30 minutes befre additin f carbachl, a time sufficient t prduce steady-state changes in the actin ptential rate f rise and duratin, respectively, by the channel-blcking cmpunds. Effect f Changes in Extracellular Inic Cnstituents In initial experiments, 20 mm Cs + was added t the Tyrde's slutin t blck resting K + cnductance (g K1 ). As described by thers, 28 the current-vltage relatin became linear (frm 100 t 40 mv) in the presence f Cs + (data nt shwn), and R^, increased t 13.1 ± 2.8 KCl (n = 7) frm an initial value f 7.0 ± 1.8 Kft (/7<0.01). The membrane deplarized t - 57 ± 4.0 mv because f the Cs + -induced blck f K + current (Table 2). At this initial value f the membrane ptential, the deplarizatin by carbachl (4.1 ±0.5 mv) was nt significantly different (p>0.1) frm that bserved befre Cs + (3.8 ±0.3 mv). When the initial resting V m in Cs + was adjusted (cmpensated) t the value bserved befre Cs + ( 79 mv), the deplarizatin evked by carbachl was larger (Table 2). These results cnfirm the cnclusin suggested abve that Table 1. Resting Ptential Changes by Carbachl (10~ 4 M) in Pertussis-Txin-Treated and Cntrl Atrial Cells Resting ptential (mv) Treatment Initial + Carbachl A* E rcv (mv) Saline (n = 5) -77±2.ii -81: t ±2.3 Pertussis txin -76±l.ii -71: ,0±0.7 9 *Membrane hyperplarizatin indicated by minus sign, membrane deplarizatin by plus sign.

5 440 Circulatin Research Vl 61, N 3, September 1987 Table 2. Effect f Cs + (20 mm) n Carbachl (10~ 4 M)- Induced Deplarizatin* Befre Cs + In Cs + Uncmpensated Cmpensatedt Initial resting ptential (mv) ± Deplarizatin amplitude (mv) 3.8± ± ±0.8t *n = 1 cells examined in each cnditin; all cells are frm pertussis-txin-treated animals. trefers t cnditin where initial resting ptential was adjusted t value bserved in the absence f Cs + befre supervisin with carbachl. tp<0.005 when cmpared with deplarizatin in Cs + (uncmpensated). carbachl-induced deplarizatin did nt arise frm a reductin f K + cnductance. A similar result was btained in the presence f 0.2 mm Ba 2+ where carbachl retained its ability t deplarize the membrane. In the presence f 20 mm Cs +, resting K + channels (i K i) activated by membrane vltage and K + channels activated by muscarinic agnists are blcked Additinally, the presence f pertussis txin prevents the pening f K + channels by muscarinic agnist. 7 " 9 In the presence f 20 mm Cs + and treatment with pertussis txin, carbachl (10~ 4 M) decreased R^, frm an initial value f 13.1 ±2.8 t 8.2±1.7 Kfl (n = 7, p<0.0\) when it deplarized atrial cells. If carbachl were smehw able t increase K + cnductance despite the presence f Cs + and pertussis txin treatment, membrane hyperplarizatin rather than deplarizatin shuld have ccurred. The bservatin that carbachl deplarized the membrane under these cnditins indicated that the agnist might increase resting Na + cnductance. The suppsitin was tested in a series f experiments in which the extracellular Na + cncentratin ([Na + ]J was reduced by replacement with istnic sucrse. Carbachl (10~ 4 M) deplarized atrial membranes 3.7 ±0.3 mv (n = 24) in 150 mm [Na + ] 0. The amplitude f the deplarizatin averaged 87 ± 9% f initial value in 75 mm [Na + ] 0 (n = 8); 61 ± 7% f initial value in 37.5 mm [Na + ] (n= 10);and53±8%finitialvaluein 13 mm [Na + ] 0 (/i =11). The dependence n extracellular sdium cncentratin is cnsistent with the hypthesis that Na + cnductance increased when carbachl deplarized. In the experiments with sucrse, [C\'] was reduced alng with [Na + ]. Frm measurements f Cl" distributin in frg ventricle, it was nt pssible t rule ut active C\~ transprt althugh there is n evidence fr such transprt. 29 If the [Cl"], were greater than that expected frm passive distributin in avian atrial muscle, the pssibility culd nt be excluded that carbachl deplarized by increasing Cl" efflux. Mrever, the inic strength f the bathing slutin decreased when sucrse replaced NaCl, thereby mdifying the activities f ther ins and making it mre difficult t assess the eventual cntributins f ins such as K + and Ca 2+ t the deplarizatin. In rder t avid these prblems, an attempt was made t duplicate these experiments with chline chlride as the replacement fr NaCl. Hwever, when 75 mm chline chlride replaced 75 mm NaCl, the carbachl-induced deplarizatin was eliminated. The mst likely explanatin is that chline acts as an antagnist f carbachl at muscarinic receptrs. The IC 50 (cncentratin t displace 50% f bund ligand) f chline needed t displace the muscarinic antagnist ligand, 3 H-quinuclidinyl benzilate, frm binding sites in brain was 1.8±0.6 mm. 30 Our experiments were dne at mre than 30 times this cncentratin. Atrial Cntractins In atria frm saline-treated animals, carbachl evked a characteristic negative intrpic effect with a threshld cncentratin near 10~ 8 M and maximum inhibitin at abut 10~ 6 M. 9 By cntrast, carbachl evked a psitive intrpic effect in atria frm pertussis-txin-treated chicks. The threshld cncentratin was >10" 6 M fr the psitive intrpic effect. As shwn in Figure 6, carbachl (10~ 5 t 10~ 3 M) evked a graded increase in the frce f cntractins in atria frm pertussis-txin-treated chicks. The results f all experiments with carbachl are shwn in Figure 7. The psitive intrpic effect f 10" 4 M carbachl, which increased frce mre than twfld, was unchanged in the presence f prpranll (3 X 10" 7 M). Hwever, atrpine (3 x 10" 7 M) prevented carbachl frm increasing the frce f cntractin (Figure 7). Acetylchline als evked a psitive intrpic effect in atria frm pertussis-txin-treated animals. The threshld cncentratin was >10" 4 M, and frce averaged nly 113±7% (n = 5) f initial at 10" 3 M (data nt shwn). Hwever, physstigmine (10~ 6 M) permitted the detectin f a psitive intrpic effect f acetylchline at lwer cncentratins (>10 =<f 'M), and the maximum effect at 10" 3 M was 172 ± 15% f the initial value (n = 3). The negative intrpic effect f xtremrine seen in saline-treated animals (n = 3) was largely but nt cmpletely vercme by pertussis txin treatment (Figure 8). Hwever, unlike carbachl and acetylch- CONTROL CARBACHOL I 300mg Tensin IO~ 5 M IO" 4 M IO~ 3 M FIGURE 6. Psitive intrpic effect f carbachl in atrial muscle frm IAP (pertussis txin)-treated chick. Upper trace is time marker; largest signals are minute marks. Lwer trace is develped frce (calibratin, 300 mg) in atrial muscle strip pacedat3 Hz. Prpranll (3 X 10' 7 M) was present in Tyrde's slutin thrughut experiment. Intrductin f indicated cncentratins f carbachl is shwn by dwnward deflectins f time trace.

6 Tajima et al PI Hydrlysis and Muscarinic Stimulatin IAP fc I A P+ Prpfnll l I A P + Prprnll -f Atrpine (n = 3) < 0 3xlO" 7 3xlO" 6 3xlO" 5 SxlO" 4 3xlO~ L C 10"' IO" b 10" CARBACHOL(M) FIGURE 7. Summary f cntractin experiments with carbachl in atria frm lap-treated chicks. Ordinate: frce f cntractin as % initial (= 100%); abscissa: mlar carbachl cncentratin. Psitive intrpic effect f carbachl was same in IAP alne (circles) and in IAP plus 3 X 10' 7 M prpranll (triangles). Hwever, additin f 3 X 10~ 7 M atrpine t latter slutin (squares) was assciated with cmplete blckade f psitive intrpic effect f carbachl. line, xtremrine was nt able t increase the frce f cntractin in atria frm pertussis-txin-treated animals, even at 10~ 4 M. Phsphinsitide Hydrlysis The ability f muscarinic agnists t prmte [ 3 H]InsP frmatin was examined in chick atria. As shwn in Figure 9, carbachl stimulated phsphin- 2 h- u <x <r t- z u I0" 9 IO~ 8 I0" 7 I0" 6 OXOTREMORINE FIGURE 8. Blckade f negative intrpic effect f xtremrine by IAP treatment. Ordinate: frce f cntractin as % initial ( = 100%); abscissa: mlar cncentratin f xtremrine. In saline-treated animals (pen circles), xtremrine exerted significant negative intrpic effect at 10'" M; cntractins culd nt be evked at >/0" M. In lap-treated animals (slid circles), negative intrpic effect was largely ( 75%) but nt cmpletely prevented. All tissues were paced at 3 Hz. (M) 10" 10" 0 3xlO" B 3xlO" 7 3xlO" 6 3x10-= 3x10-* lagnist] FIGURE 9. Agnist-induced stimulatin f 'H-insitl phsphate (['HJInsP) prductin in chick atria. Ordinate: ['HJInsP in cpmlmg prtein; abscissa: carbachl r xtremrine was added t labelled chick atria in presence f 10 mm LiCI at cncentratins indicated. Unstimulated atria (n agnist) received nly 10 mm LiCI. Values shwn are means±sem (n = 3) frm representative experiment. sitide hydrlysis in a cncentratin-dependent fashin. The threshld cncentratin was 10~ 6 M, and [ 3 H]InsP frmatin was maximal at >3x 10~ 4 M. There was little r n effect f xtremrine n phsphinsitide hydrlysis, even at cncentratins as great as 3 x 10~ 4 M (Figure 9). Acetylchline at 10~ 3 M gave apprximately half the respnse elicited by a maximal cncentratin f carbachl, and the respnse t carbachl was inhibited by 10" 6 M atrpine (Table 3) but nt by curare (data nt shwn). Verapamil did nt significantly decrease carbachl-stimulated [ 3 H]InsP frmatin (nt shwn), a finding cnsistent with the lack f effect f this Ca 2+ channel blcker n the carbachl-induced deplarizatin. Carbachl-induced stimulatin f [ 3 H]InsP frmatin was unchanged in atria frm pertussis-txin Table 3. Effects f Chlinergic Agnists and Antagnists n Phsphinsitide Hydrlysis Cntrl Carbachl (lo" 3 M) Acetylchline (10~ 3 M) Carbachl (10" 3 M) + atrpine (IO~" 6 M) [ 3 H]InsP (cpm/mg prtein) 110±12 324± it 12 Data are means ± SEM (n = 6). Acetylchline was tested in the presence f 10~ 6 M physstigmine.

7 442 Circulatin Research Vl 61, N 3, September 1987 treated chicks (Figure 10). This finding is cnsistent with bservatins made n embrynic chick heart cells. 18 In atria frm saline-treated chicks (n = 8), carbachl increased [ 3 H]InsP by ~450 cpm/mg prtein abve the basal [ 3 H]IP level (~furfld). In atria frm pertussis-txin-treated chicks (n = 9), the basal [ 3 H]InsP was smewhat higher, but the increase elicited by carbachl (~475 cpm) was nearly the same as that in untreated tissue (Figure 10). The maximal frmatin f [ 3 H]InsP by carbachl was nt significantly different (p = 0.4) in the absence and presence f pertussis txin treatment. Discussin In atria frm pertussis-txin-treated chicks, carbachl increased the hydrlysis f phsphinsitides, deplarized the membrane, and exerted a psitive intrpic effect. These phenmena shared several characteristic pharmaclgic features that included 1) cncentratin dependence, 2) agnist selectivity, 3) antagnist sensitivity, and 4) resistance t blckade by pertussis txin. The cncentratins f carbachl required t hydrlyze phsphinsitides, deplarize the membrane, and increase the frce f cntractin in atria ranged frm S:10~ 6 M t 10" 3 M. This cncentratin range is different frm that (10~ 8 t 10" 6 M) required t increase K + cnductance, 9 t decrease Ca 2+ cnductance, 9 and t inhibit isprterenl-stimulated camp accumulatin in chick heart. Acetylchline was an agnist fr all three respnses, althugh it was less efficacius than carbachl. Oxtremrine did nt prmte phsphinsitide hydrlysis, nr did it deplarize the membrane r increase the frce f cntractin. Atrpine blcked the ability f carbachl t stimulate phsphinsitide hydrlysis, deplarize the membrane, r increase the frce f cntractin. Prpranll had n effect n the deplarizatin and psitive intrpic actin f carbachl, and neither prpranll nr _ 800 at a 600 e E -H re n I W/// W/A Cnt Carb - Pertussis txin T 1 Cnt Carb + Pertussis txin FIGURE 10. Pertussis txin des nt blck carbachl-induced stimulatin flnsp prductin. Atria frm chicks treated r nt treated with 1.5 fig pertussis txin were labelled and then incubated with vehicle (10 mm LiCl cntrl) r carbachl (1 mm) fr 30 minutes. Ordinate: [ 3 H]InsP in cpm/mg prtein. Values shwn are mean±sem (n = 4-5). prazsin antagnized the increase in InsP accumulatin prduced by carbachl. These results indicate that deplarizatin, the psitive intrpic respnse, and PI hydrlysis are all mediated via muscarinic receptrs and make it unlikely that release f endgenus nrepinephrine by carbachl is invlved. The same pharmaclgic prfile is seen in cat papillary muscle. 32 In this preparatin, carbachl als increased phsphinsitide hydrlysis and the frce f cntractin, whereas xtremrine was withut effect n either phenmenn. Mrever, atrpine but nt phenxybenzamine, prpranll, r hexamethnium ppsed the stimulatin f phsphinsitide hydrlysis and the psitive intrpic effect f carbachl in this preparatin. 32 The psitive intrpic effects f ACh in cardiac Purkinje fibers 33 and f carbachl n guinea pig papillary muscle 21 were als blcked by atrpine but nt by prpranll, phentlamine, hexamethnium, r verapamil. Pertussis txin treatment des nt blck the increased phsphinsitide hydrlysis, membrane deplarizatin, r the psitive intrpic effect f carbachl in chick atrial tissue Under these cnditins f pertussis txin treatment, >90% f the labelling f guanine nucletide binding prteins Gi and G is eliminated, indicating that mre than 90% f these G prteins are ribsylated. 16 Thus, if either f these prteins is invlved as a transducer fr the stimulant effects f carbachl in atria, it must be that 5-10% f G^GJ is adequate t transduce these effects. Hwever, under these cnditins f pertussis txin treatment, 9 it has been reprted that the affinity f muscarinic receptrs (machr) fr carbachl is decreased, muscarinic inhibitin f adenylate cyclase is virtually ablished, and the hyperplarizatin resulting frm increased K + cnductance is inhibited. These data argue that the link between machr and cellular effectrs respnsible fr the stimulant actins f carbachl is nt a pertussis-txin-sensitive prtein (G, r G ) and that neither inhibitin f adenylate cyclase nr activatin f K + cnductance is invlved. It is pssible that an as yet unidentified G prtein serves as a transducer fr the stimulant actins f carbachl. Muscarinic receptr mediated activatin f phsphlipase C in muse excrine pancreas cells and the stimulatin f IP 3 frmatin caused by carbachl depend n guanine nucletides. 34 The "G" prtein transducer f this muscarinic effect is nt inhibited by pretreatment with either pertussis txin r chlera txin. In embrynic chick heart cells permeabilized with sapnin, the frmatin f [ 3 H]insitl phsphates is als stimulated by guanine nucletides (manuscript in preparatin). 35 Since the PI respnse in the chick heart cells is nt blcked by either pertussis txin r chlera txin,' 8-35 it appears that a nvel G prtein may regulate PI hydrlysis in the mycardium, as suggested fr ther cells The present study has btained pharmaclgic evidence cnsistent with the cncept that the increased hydrlysis f phsphinsitides, membrane deplarizatin, and psitive intrpic effect are mediated

8 Tajima et al PI Hydrlysis and Muscarinic Stimulatin 443 thrugh a cmmn pathway and may be casually related t each ther. One hypthesis fr a causal relatin amng these respnses is that certain muscarinic agnists increase insitl trisphsphate (IP 3 ) cncentratin within the cell. As a secnd messenger, IP 3 wuld release Ca 2+ frm sarcplasmic reticulum (SR) stres. Indeed, in chick heart cells, IP 3 is increased within 10 secnds after the additin f carbachl. 35 Furthermre, IP 3 (^30 /xm) has been shwn t induce the release f Ca 2+ frm the SR f mechanically skinned rat ventricular cells. 36 Even at 200 /xm, IP 3 had n effect either n mitchndria r n myfilaments in this preparatin. Althugh Ca 2+ release frm SR by IP 3 was judged incmpatible with a rle in excitatin-cntractin cupling in heart muscle, 36 the ability f IP 3 t increase frce develpment by releasing Ca 2+ is cnsistent with ur hypthesis that it culd serve as a secnd messenger in the psitive intrpic effect f carbachl. An alternative hypthesis, still invlving the PI respnse as a primary mediatr, wuld be that prtein kinase C is activated because f the frmatin f diacylglycerl. Hypthetically, prtein kinase C culd phsphrylate and thereby activate plasma membrane in channels, r it culd phsphrylate and alter the calcium sensitivity f the myfilaments. Amng the variety f cardiac membrane and myfibrillar prteins shwn t be substrates fr prtein kinase C 35 is a 15 kda plasma membrane prtein that may be invlved in vltage-sensitive Ca 2+ channels The pssibility that the psitive intrpic effect f muscarinic agnists arises frm increased entry f Ca 2+ thrugh vltage-sensitive channels seems remte. In canine cardiac Purkinje fibers, acetylchline evked an atrpine-sensitive psitive intrpic effect at cncentratins frm 10~ 8 t 10" 4 M." Acetylchline als increased actin ptential duratin at 50% (5.4 mm K + -Tyrde's slutin) and restred Ca 2+ -dependent actin ptentials t fibers deplarized by 22 mm [K + ] but nly at cncentratins ^10" 5 M. Hwever, tw bservatins mil itated against the view that the psitive intrpic effect f acetylchline arse frm increased Ca 2+ entry thrugh vltage-sensitive channels. 33 First, the psitive intrpic effect culd be detected at acetylchline cncentratins that had n effect n actin ptential duratin at 50% and did nt restre Ca 2+ -dependent actin ptentials. Secnd, the psitive intrpic effect was nt antagnized by verapamil. The psitive intrpic effect was independent f catechlamine release. Indeed, it was suggested that the psitive intrpic effect f acetylchline in Purkinje fibers was similar under certain cnditins t that seen in wrking mycardium." In sheep cardiac Purkinje fibers, acetylchline increased actin ptential duratin at cncentratins as lw as 10" 7 M This effect, which was attributed t a reductin f inward backgrund currents, 41 was prevented by atrpine and was independent f endgenus catechlamines Because this actin f acetylchline seemed specific fr sheep cardiac Purkinje fibers, 3940 its applicability t the psitive intrpic effect f muscarinic agnists in ther cardiac tissues is unknwn. Our experimental results 16 in avian atrium and thse f thers 21 in mammalian ventricle indicate that carbachl and acetylchline d nt increase vltagedependent Ca 2+ entry since neither the plateau amplitude nr the actin ptential duratin was significantly increased when the psitive intrpic effect ccurred. That the membrane deplarizatin culd arise frm an effect f increased intracellular Ca 2+ (rather than frm Ca 2+ entry thrugh vltage-sensitive channels) is suggested by experiments in sheep cardiac Purkinje fibers. Strphanthidin induces transient deplarizatins that are generated by a transient inward current carried primarily by Na It was prpsed that this current develps because strphanthidin inhibits the Na + pump and elevates intracellular Ca 2+, which then activates a passive plasma membrane channel that admits Na + and K In ur hypthesis, elevated intracel lular Ca 2 + released by IP 3 actin n the SR culd serve a similar functin. The elevated intracellular Ca 2+ wuld activate a plasma membrane channel that admits Na + and thereby deplarizes the membrane. Neither the carbachl-induced deplarizatin f atrial fibers (present reprt) nr the strphanthidin-induced transient deplarizatin 25 is directly sensitive t TTX r verapamil, blckers f vltage-dependent Na + and Ca 2+ channels, respectively. In Purkinje fibers that are either electrically stimulated r vltage-clamped s as t increase Na + and Ca + entry thrugh vltagedependent channels, TTX and Ca 2+ channel blckers can diminish transient inward currents indirectly by virtue f having diminished Na + and Ca 2+ currents, respectively In guinea pig papillary muscle, a different hypthesis has been advanced by Krth and Kiihlkamp 2 ' whse findings are very similar t urs. They als demnstrated an atrpine-sensitive psitive intrpic effect f carbachl. The respnse was assciated with an increased intracellular activity f Na + that was nt prevented by TTX and was attributed t muscarinic agnist increasing Na + permeability f the plasma membrane. 21 Mre recently, these investigatrs have reprted that carbachl was the mst ptent and xtremrine was the least ptent muscarinic agnist fr evking a psitive intrpic effect in guinea pig ventricle. 43 Acetylchline, which increased intracellular Na + activity by half as much as carbachl, was als half as effective as carbachl in prducing a psitive intrpic effect. In cntrast t the results with all ther muscarinic agnists tested (carbachl, methachline, acetylchline, and bethanecl), atrpine did nt prevent the psitive intrpic effect f xtremrine. Mrever, stimulatin f PI hydrlysis with eventual mdulatin f catin fluxes was suggested as a pssible mechanism fr the psitive intrpic effect f chline esters. 43 The mechanism by which Na + permeability increased is nt clear but again culd invlve a prtein-kinase-c-mediated phsphrylatin. Where their hypthesis differs frm urs is Krth and Kiihlkamp's prpsal that the entry f Na + was the initiating

9 444 Circulatin Research Vl 61, N 3, September 1987 event that then brught abut a psitive intrpic effect by stimulatin f Na-Ca exchange. (Indeed, a similar mechanism had riginally been cnsidered as a pssible explanatin fr the initiatin f a transient inward current by digitalis glycsides, 25 but these authrs fund this mechanism less satisfactry than did thers. 26 ) The carbachl-induced increase f intracellular Na + was greatly diminished when [Ca 2+ ] was 1.6 mm. 2 ' This finding is puzzling, fr the initial entry f Na + shuld nt be changed when [Ca 2+ ] 0 is reduced unless there is sme essential but as yet unknwn effect f [Ca 2+ ] n the reactin mechanism. 21 Reslutin f whether Na + permeability increases secndary t r leads t increases in Ca 2+ will require experiments with islated mycytes under space clamp cnditins mre ptimal than thse available in multicellular preparatins. In any event, the hypthesis that carbachl affects Na + rather than K + permeability is well supprted by tw findings. First, muscarinic agnist increased membrane cnductance in atrial preparatins treated with pertussis txin and Cs + sufficient t blck drug-induced changes f K + cnductance. Secnd, the carbachl-induced deplarizatin was directly prprtinal t extracellular Na +. The stimulant effects f carbachl in chick atria cells (deplarizatin and psitive intrpy) are the ppsite f thse (hyperplarizatin and negative intrpy) mediated by inhibitry guanine nucletide binding prteins G i and G. There is reasn t think that such stimulant actins are present, but masked, when G ; and G develp and becme fully functinal. In very yung (4th incubatin day) embrynic hearts, 27 acetylchline and carbachl deplarized the membranes f sinatrial cells. The deplarizatin, like that described in the present reprt, was diminished in the absence f extracellular Na +27 and resistant t blckade by TTX. 44 The ability f acetylchline and carbachl t deplarize the sinatrial membrane in 4-day embrynic hearts was related nt nly t the increased Na + entry but als t the fact that the increase f K + cnductance by these muscarinic agnists was nt well develped. (An increase f G^ and G ccurred between the 4th and 8th incubatin days in embrynic chick atria, 45 and this phenmenn was assciated with an increased sensitivity f the tissue t the negative chrntrpic effect f carbachl, 45 which had been attributed t an increased activatin f K + channels by muscarinic agnist. 27 ) The present experiments carried ut with atria frm newbrn chicks bear a strng similarity insfar as the deplarizatin prduced by carbachl and acetylchline becmes evident when treatment with pertussis txin prevents the increased K + cnductance. The previus electrphysilgic and bichemical 31 findings in the embrynic heart prvide additinal evidence fr the phsphinsitide hypthesis advanced in this reprt. Carbachl (10~ 5 t 10~ 3 M) increased [ 3 H]InsP frmatin in embrynic chick hearts between the 4th and 13th incubatin days. Interestingly, the greatest stimulatin (eightfld increase) was detected n the 4th incubatin day; the stimulant effect declined mntnically t a twfld increase n the 13th incubatin day. 31 In summary, stimulant effects f muscarinic agnists have been detected in chick atrial cells that appear t be independent f the guanine nucletide binding prteins, G, and G, and t be mediated thrugh mechanisms ther than inhibitin f adenylate cyclase r activatin f K + cnductance. Our findings have been incrprated int a hypthesis in which the stimulatry effect f muscarinic agnists n the mycardium is secndary t increased PI turnver. One pssible mechanism fr this effect wuld be that IP 3 is the intracellular messenger fr the eventual membrane deplarizatin and psitive intrpic effects bserved. Alternatively, activatin f prtein kinase C might affect sarclemmal in channels. The precise inic mechanism fr the deplarizatin and psitive intrpic effect cannt be decided n the basis f present findings. Either an activated intracellular calciumdependent passive in transprt reactin r an altered sdium-calcium exchange wuld prvide a nvel mechanism fr the stimulant effects f parasympathetic transmitter. References 1. Lffelhlz K, Pappan AJ: The parasympathetic neureffectr junctin f the heart. Pharmacl Rev 1985;37:l Reuter H: Calcium channel mdulatin by neurtransmitters, enzymes and drugs. Nature 1983;301: Watanabe AM: Chlinergic agnists and antagnists, in Rsen MR, Hffman BF (eds): Cardiac Therapy. Bstn, Martinus Nijhff, 1983, pp Hescheler J, Kameyama M, Trautwein W: On the mechanism f muscarinic inhibitin f the cardiac Ca current. Pflugers Arch 1986;407: Hartzell HC, Fischmeister R: Oppsite effects f cyclic GMP and cyclic AMP n Ca 2 + current in single heart cells. Nature 1986;323: Breitwieser GE, Szab G: Uncupling f cardiac muscarinic and /3-adrenergic receptrs frm in channels by a guanine nucletide analgue. Nature 1985;317: Endh M, Maruyama M, Iijima T: Attenuatin f muscarinic chlinergic inhibitin by islet-activating prtein in the heart. Am J Physil 1985;249(Weart Circ Physil I8).H3O9-H32O 8. Pfaffinger PJ, Martin JM, Hunter DD, Nathansn NM, Hille B: GTP-binding prteins cuple cardiac muscarinic receptrs t a K channel. Nature 1985;317: Srta S, Tsuji Y, Tajima T, Pappan AJ: Pertussis txin treatment blcks hyperplarizatin by muscarinic agnists in chick atrium. Circ Res 1985;57: Yatani A, Cdina J, Brwn AM, Birnbaumer L: Direct activatin f mammalian atrial muscarinic ptassium channels by GTP regulatry prtein G v. Science 1987;235: Lgthetis DE, Kurachi Y, Galper J, Neer EJ, Clapham DE: The fiy subunits f GTP-binding prteins activate the muscarinic K + channel in heart. Nature 1987;325: Sakmann B, Nma A, Trautwein W: Acetylchline activatin f single muscarinic K + channels in islated pacemaker cells f the mammalian heart. Nature 1983;3O3:25O Sejima M, Nma A: Mde f regulatin f the ACh-sensitive K-channel by the muscarinic receptr in rabbit atrial cells. Pflugers Arch 1984;400: Iijima T, Irisawa H, Kameyama M: Membrane currents and their mdificatin by acetylchline in islated single atrial cells f the guinea pig. J Physil (Lnd) 1985;359: Carmeliet E, Mubagwa K: Characterizatin f the acetylchline-induced ptassium current in rabbit cardiac Purkinje fibres. J Physil (Lnd) 1986;371: Srta S, Tsuji Y, Pappan AJ: The psitive intrpic effect f

10 Tajima el al PI Hydrlysis and Muscarinic Stimulatin 445 muscarinic agnists in pertussis txin-treated chicks (abstract). Circulatin!985;72(suppl 1II):I1I Brwn JH, Brwn SL: Agnists differentiate muscarinic receptrs that inhibit cyclic AMP frmatin frm thse that stimulate phsphinsitide metablism. J Bil Chem 1984; 259: Masters SB, Martin MM, Harden TK, Brwn JH: Pertussis txin des nt inhibit muscarinic receptr-mediated phsphinsitide hydrlysis r calcium mbilizatin. Bichem J 1985;227: Inue D, Hachisu M, Pappan AJ: Acetylchline increases resting membrane ptassium cnductance in atrial but nt in ventricular muscle during muscarinic inhibitin f Ca 2+ - dependent actin ptentials in chick heart. Circ Res 1983; 53: Bredikis J, Bukauskas F, Veteikis R: Decreased intercellular cupling after prlnged rapid stimulatin in rabbit atrial muscle. Circ Res 1981;49: Krth M, Kuhlkamp V: Muscarinic receptr-mediated increase f intracellular Na + -in activity and frce f cntractin. PflugersArch 1985;403: Higgins D, Pappan AJ: Develpmental changes in the sensitivity f the chick embry ventricle t /3-adrenergic agnist during adrenergic innervatin. Circ Res 1981 ;48: Masters SB, Quinn MT, Brwn JH: Agnist induced desensitizatin f muscarinic receptr mediated calcium efflux withut cncmitant desensitizatin f phsphinsitide hydrlysis. Ml Pharmacl l985;27: Bradfrd M: Quantitatin f micrgram quantities f prtein utilizing the principle f prtein-dye binding. Anal Bichem 1976;72: KassRS.TsienRW, WeingartR: Inic basis f transient inward current induced by strphanthidin in cardiac Purkinje fibres. J Physil (Lnd) 1978;281: Arlck P, Katzung BG: Effects f sdium substitutes n transient inward current and tensin in guinea-pig and ferret papillary muscle. J Physil (Lnd) I985;360: Pappan AJ: Sdium-dependent deplarizatin f nn-innervated embrynic chick heart by acetylchline. J Pharmacl ExpTher 1972; 180: OjedaC, RugierO,TurneurY: EffectsfCs n acetylchline induced current. PflugersArch 1981;391: Macchia DD, Plimeni PI, Page E: Cellular Cl cntent and cncentratin f amphibian skeletal and heart muscle. Am J Physil I978;235:C122-C Haubrich DR, Risley EA, Williams M: Effects f deanl, chline and its metablites n binding f 'H] quinuclidinyl benzilate t rat brain membranes. Bichem Pharmacl l979;28: Orellana S, Brwn JH: Stimulatin f phsphinsitide hydrlysis and inhibitin f cyclic AMP frmatin by muscarinic agnists in develping chick heart. Bichem Pharmacl 1985;34:I Gjrstrup P. Harding H, Jacbssn B, Ransnas L: On the psitive intrpic respnse f muscarinic receptr stimulatin in feline papillary muscle in vitr (abstract). J Physil (Lnd) 1985;369:84P 33. Gilmur RF Jr, Zipes DP: Psitive intrpic effect f acetylchline in canine cardiac Purkinje fibers. Am J Physil \985;249(Heart Circ Physil 18):H735-H Merritt JE, Taylr CW, Rubin RW, Putney JW Jr: Evidence suggesting that a nvel guanine nucletide regulatry prtein cuples receptrs t phsphlipase C in excrine pancreas. Bichem J 1986;236: Brwn JH, Jnes LG: Phsphinsitide metablism in the heart, in Putney J (ed): Phsphinsitides and Receptr Mechanisms. New Yrk, Alan R Liss Inc, 1986, pp Fabiat A: Insitl (l,4,5)-trisphsphate-induced release f Ca 2+ frm the sarcplasmic reticulum f skinned cardiac cells (abstract). Biphys J 1986;49:190a 37. Presti CF, Sctt BT, Jnes LR: Identificatin f an endgenus prtein kinase C activity and its intrinsic 15-kiIdaltan substrate in purified canine cardiac sarclemmal vesicles. J Bil Chem 1985;260: LindemanJP: a-adrenergic stimulatin f sarclemmal prtein phsphrylatin and slw respnses in intact mycardium. J Bil Chem 1986;261: Lipsius SL, Gibbns WR: Acetylchline lengthens actin ptentials f sheep cardiac Purkinje fibers. Am J Physil 1980;238(tfea/7 Circ Physil 7):H237-H Carmeliet E, Ramn J: Electrphysilgical effects f acetylchline in sheep cardiac Purkinje fibers. Pflugers Arch 1980; 387: Carmeliet E, Ramn J: Effect f acetylchline n timeindependent currents in sheep cardiac Purkinje fibers. Pflugers Arch 1980;387: Kass RS, Lederer WJ, Tsien RW, Weingart R: Rle f calcium ins in transient inward currents and aftercntractins induced by strphanthidin in cardiac Purkinje fibres. J Physil (Lnd) 1978;281: Krth M, Kuhlkamp V: Muscarinic receptrs mediate negative and psitive intrpic effects in mammalian ventricular mycardium: differentiatin by agnists. BrJ Pharmacl 1987;90: Pappan AJ: Pharmaclgy f Heart Cells During Ontgenesis, in Bianchi CP, Naranashi T (eds): Advances in General and Cellular Pharmaclgy. New Yrk, Plenum Publishing C, 1976, vl 1, pp Halvrsen SW, Nathansn NM: Ontgenesis f physilgical respnsiveness and guanine nucletide sensitivity f cardiac muscarinic receptrs during chick embrynic develpment. Bichemistry 1984;23: KEY WORDS muscarinic agnist deplarizatin psitive intrpic effect phsphlipase C insitl trisphsphate atrial membrane pertussis txin prtein kinase C